Field of the Invention
The present invention generally relates to pharmaceutical compositions and oncological treatment methods. In particular, the invention provides improved compositions for targeting chemotherapeutics to mitochondria, and methods for selective therapy of mammalian cancers, and in particular, human cancers such as gliomas.
Description of Related Art
Gliomas: Prognosis and Treatment. Nearly 10,000 Americans each year are diagnosed with malignant glioma. Of those, 50% survive one year, and only 20% survive two years. Five-year survival rate is <3%. Conventional treatment consists of a triad: surgery (if the location allows it), radiotherapy, and chemotherapy. After surgery, chemotherapy (normally in the form of DNA acylating agents such as temozolomide or carmustine or more rarely, the topoisomerase inhibitor, irinotecan) is initiated. In certain patients, carmustine may also be delivered in the form of wafers placed into the post-surgical wound.
Gliomas are the most common malignant brain tumors reported in humans. Gliomas are neuronal malignancies that arise from an uncontrolled proliferating cell of the central nervous system. Patients diagnosed with gliomal cancer have a dismal prognosis, and although symptoms vary with the particular site of the tumor, they tend to develop very quickly due to the rapid growth behavior of the tumor cell. Gliomas can originate from several cell types including ependymal cells, astrocytes, oligodendrocytes and different types of glia cells. Clinically, gliomas are divided into four grades, which are determined by pathologic evaluation of the tumor. Low-grade gliomas are well-differentiated and slower growing, thus biologically less aggressive, and therefore offer a relatively better prognosis for the patient. Conversely, high-grade gliomas are anaplastic, fast-growing, and invasive towards adjacent tissues. Consequently, high-grade gliomas offer a worse prognosis for the patient. Unfortunately, the most aggressive of these grades, grade 4 or glioblastoma multiforme (GBM), is also the most frequent in humans. Because most patients with GBMs die of their disease in less than a year (and essentially no GBM patient has what would be considered a “long-term survival”), the development of more effective treatment regimens for the disease has been vigorously pursued for more than fifty years, with, unfortunately, only limited success to date.
Cancers, Mitochondria and Hydrogen Peroxide as a Mitogen. Hydrogen peroxide is a product of mitochondrial respiration, which produces superoxide by the one-electron reduction of molecular oxygen with hydrogen; peroxide is then generated by the action of superoxide dismutase or due to spontaneous dismutation (Vizi, 2000; Boveris and Chance, 1973). Hydrogen peroxide is a potent mitogen, particularly in microglia (Jekabsone et al., 2006; Mander et al., 2006). Cancer cells produce high amounts of hydrogen peroxide, which is linked to key alterations in cancer, including cell proliferation, apoptosis resistance, metastasis, angiogenesis and hypoxia-inducible factor 1 activation (Droge, 2002). In the absence of mitochondrially generated hydrogen peroxide, many cancers upregulate other enzymes, which produce hydrogen peroxide as a byproduct of their function. One such enzyme is monoamine oxidase.
Monoamine Oxidase A (MAO-A) and Monoamine Oxidase B (MAO-B)-Substrate and Inhibitor Specificity. Monoamine oxidases are the major enzymes used by the body to metabolize monoamine hormones and neurotransmitters such as epinephrine, norepinephrine, serotonin, and dopamine. There are two MAO subtypes, MAO-A and MAO-B, which are encoded by two separate genes but have a high degree of homology, and are both localized to the outer mitochondrial membrane. The two enzymes have different substrate/inhibitor specificity and tissue localization in different mammals.
Endogenous Substrates. MAO-A oxidatively deaminates epinephrine, norepinephrine, and serotonin and is found in the brain in adrenergic and noradrenergic neurons.
MAO-B acts preferentially on phenylethylamine and tele-methylhistamine as substrates and is present in astrocytes and in serotoninergic and histaminergic neurons.
Inhibitors. In addition to different substrate specificities, the two MAO's also have different specificities toward inhibitors. MAO-A is classically inhibited by clorgiline, and MAO-B is classically inhibited by L-deprenyl (most often called selegiline).
The major structural difference between human MAO-A and B is that MAO-A has a single ovoid substrate cavity of ˜550 Å3 in volume and MAO-B contains an hour-glass-shaped cavity with an upper volume of ˜290 Å3, and a lower substrate cavity of ˜400 Å3 (Milczek et al., 2011).
The differential sensitivity toward inhibitors is a function of the differences in the structure of the two enzymes. The amino acid sequences of human MAO-A (red) and MAO-B (blue) are shown in FIG. 1.
The presence of a proline residue in position 102 of the B structure, with respect to the corresponding alanine in position 111 of the A structure, causes a change in the loop formed by the following 19 amino acids. The difference between the position and nature of the two loops cause the entrance to the reactive pocket in MAO-B to be smaller, and more hydrophobic than is the case in MAO-A. One consequence is that larger, bulkier, groups are able to enter the pocket of MAO-A.
The difference in the substrate channels, leading to the FAD active site pocket, of the two enzymes is shown in FIG. 2. This figure draws upon a number of crystal structures of human MAO-A and MAO-B, where the enzymes were pretreated with specific inhibitors. Overlaying structures indicates which amino acid variants between the two enzymes are responsible for the different substrate and inhibitor specificities. Geha et al. (2001) demonstrated that the substrate/inhibitor specificities of the two forms of the human MAO could be inverted by double substitutions of hMAO-A-I335Y and hMAO-B-Y326I indicate that the large, aromatic, residues midway along the MAO channels largely control substrate specificity.
Monoamine Oxidase B in Glioma. MAO-B catalyzes deamination of dopamine through a two-electron reduction of oxygen to hydrogen peroxide. In the brains of primates and mice, it is found only in the glia and dopaminergic neurons. The activity of MAO-B is four-fold greater in glioblastoma multiforme, low-grade astrocytomas, and in anaplastic astrocytomas than in postmortem control brains or meningiomas (Gabilondo et al., 2008). It appears that hydrogen peroxide is generated by gliomal MAO-B is part of a proliferation signal. Interestingly, in high-grade prostate cancer, there is a four-fold increase in MAO-A, and again the mitotic hydrogen peroxide signaling resulting from up-regulation may be the trigger for this increase in lethality (Flamand et al., 2010).
hMAO-Specific Inhibitors. It has been previously shown that it is possible to design and synthesize small molecular families that display highly differential inhibition constants to MAO-A and MAO-B; as an example, the inventors have drawn from Regina and co-workers in their examination of pyrroles as inhibitors of hMAO. In Table 1, six structures are shown which differ by the addition of a single CH2 group.
FIG. 5 shows the inhibition constant (Ki in μM) of MAO-A and MAO-B, and the inhibition ratio, for a series of related pyrrols. Structure #7 is a highly potent MAO-B inhibitor, but has very poor inhibition of MAO-A. Increasing the length of the chain between the phenyl ring and the tertiary amine, by a single —CH2— unit, flips the sensitivity, and Structure #21, for example, is a potent MAO-A inhibitor, but is a very poor MAO-B inhibitor.
Three-dimensional modeling demonstrates that within the MAO channel, the phenyl rings of Structure #7 and Structure #21 tend to interact with the blocking amino acids described earlier. This is graphical illustrated in FIG. 3, which shows an overlay of Structure #7 and Structure #21, where the pyrrol is hydrogen bonded to the FAD ring. In MAO-B, Structure #7 does not overlay any of the amino acids lining the channel, but in MAO-A, the phenyl group of Structure #7 abuts F208.
Conversely, in MAO-A, Structure #21 does not overlay any of the amino acids lining the channel, but in MAO-B the phenyl group of Structure #21 abuts Y323.
A similar study performed on human recombinant MAO, looking at competitive inhibition using variations of a core phenylamine structure, was performed by Fierro and co-workers. The inhibitor structures and Ki constants they found is presented in Table 2.
1-Methyl-1,2,3,6-Tetrahydropyridines. The best-known exogenous substrate of MAO-B is (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) (MPTP), which is converted to its cationic form, 1-methyl-4-phenylpyridinium (MPP30), by glial cells. MPP30  is a dopamine mimetic, and is concentrated inside dopaminergic neurons via the dopamine transporter. Inside these neurons, MPP+ undergoes a second concentrating effect; moving into their mitochondria in response to the membrane potential, ΔΨ, causing both inhibition and superoxide generation at Complex I, which can result in loss of mitochondrial function, caspase activation, dopaminergic cell apoptosis, and phenotypically, Parkinson's disease (Fukuda, 2001).
The mechanism of the conversion of MPTP to MPP30  has been elucidated in two key papers: Ottoboni et al. (1989), which examined the kinetic effects of deuteration of different positions on MPTP to understand which hydrogen atoms are abstracted by the FAD reaction center; and Shi et al. (1999), who resolved the second oxidant step in the reaction.
FIG. 5 shows how MPTP is initially oxidized by MAO, with a hydrogen atom (from the C6 position on the tetrahydropyridine) and an electron from the amine to form 1-methyl-4-phenyl-2,3-dihydropyridin-1-ium (MPDP+). The MPDP+ diffuses from the enzyme and is then typically oxidized further by the mitochondrial ubiquinone pool to MPP+, although direct oxidation by aqueous molecular oxygen occurs in vitro.
Much work has been done on probing the two MAO channels using substitutions on the MPTP template [see Palmer et al. (1997) and Palmer (1998)]. She examined the kinetics of human MAO-A and bovine MAO-B (which is very similar to human) enzymes with substitutions in the phenyl ring of MPTP. FIG. 6 shows the effects of nine different substituents in the 2′-position of the phenyl ring of MPTP, and how it effects the kinetics of the two enzymes. Modeling the shape of the nine molecules shows that there is a trend in the space-filling shape of the substrate and how well it acts as a substrate. In MPTP derivatives where the rings sit in a crossed position, 90° to one another, then the molecule serves as a better MAO-A substrate, whereas flat, planar MPTP molecules are far better MAO-B substrates.
It is evident that tetrahydropyridines can be designed so that they serve as very good MAO-B substrates, but very poor MAO-A substrates. It follows that following oxidation of the tetrahydropyridine to the pyridinium cation, the now-charged molecule will partition from near the outer mitochondrial membrane, into the inside of the mitochondria, with accumulation driven by the mitochondrial membrane potential.
Amino-Propyl Ethers. In addition to their oxidation of secondary and tertiary amines, outline above, monoamine oxidases can also cleave ether or thiol ether bonds; bonds which unlike amides or ester, are generally very stable in human beings. Albers, Rawlsa and Chang demonstrated the ability of MAO-A and MAO-B to oxidize the primary or secondary amine of a propylamine ester to form an alcohol (FIG. 7).
This reaction series, amine oxidation to imine, hydration and then rearrangement converting an ether to an alcohol is biologically unusual, given that ethers are typically biologically stable. Moreover, in addition to the conversion of an ether to an alcohol, the reaction could function to cleave a thioether to a thiol or secondary/tertiary amines into primary/secondary amines.
Deficiencies in the Prior Art
One of the reasons for the resistance of GBM to therapeutic treatments is the complex character of the tumor itself. As the name GBM implies, glioblastoma is multiforme. It is multiforme both grossly (often presenting regions of necrosis and hemorrhage) and microscopically (complete with regions of pseudopalisading necrosis, pleomorphic nuclei and cells, and microvascular proliferation). Moreover, GBM is genetically diverse, with various deletions, amplifications, and point mutations leading to activation of signal transduction pathways downstream of tyrosine kinase receptors such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), as well as to disruption of cell-cycle arrest pathways by INK4a-ARF loss or by p53 mutations associated with cyclin-dependent kinase 4 (CDK4) amplification or retinoblastoma-protein (Rb) loss.
Compounding the difficulty in successful GBM treatment is the fact that surgical resection of the tumor is hampered by the topographically-diffuse nature of the tumors themselves. Moreover, the location of the GBM tumor cells within the brain can also be highly variable, resulting in the inability to completely resect this tumor. Glioma cells migrate away from the initial tumor through the brain parenchyma, collect just below the pial margin (subpial spread), surround neurons and vessels (e.g., perineuronal and perivascular satellitosis), and migrate through the white matter tracks (e.g., intrafascicular spread). As a result, the individual tumor cells diffuse over long distances, and into areas of the brain that are essential for the patient's survival. An extreme example of this behavior is a condition referred to as “gliomatosis cerebri,” in which the entire brain is diffusely infiltrated by neoplastic cells with minimal or no central focal area of tumor per se. Although gliomas do not metastasize via the bloodstream, they can spread via cerebrospinal fluid and cause what is referred to as “drop metastases” in the spinal cord. Fully one quarter of patients with GBM demonstrate multiple or multi centric GBMs at autopsy. Consequently, the infiltrative growth pattern of these tumors precludes curative neurosurgery, and high-grade gliomas almost always recur even after what was thought to be “complete” surgical resection.
Despite recent advances in therapy, treatment of malignant gliomas remains palliative. Median post-diagnosis survival for anaplastic astrocytoma is less than 3 years and for glioblastoma multiforme is typically only 12 to 14 months. Temozolomide, an oral methylating chemotherapeutic agent, became standard of care for newly diagnosed glioblastoma when used concurrently with external beam radiation followed by adjuvant therapy, although GBM continue to be highly resistant to radiation. Under even the best of circumstances (in which essentially all of the tumor can be surgically removed and the patients are fully treated with radiation and chemotherapy), the mean survival of this disease is extended only by a period of a few months.
The poor outcome of the standard treatments for GBM coupled with the diffuse nature of the disease itself, have influenced a number of attempts at novel therapeutic approaches with the aim of also killing neoplastic cells disseminated from the main tumor. To date, however, the only significant therapeutic options for GBM are limited to surgery, radiotherapy and conventional chemotherapy using drugs such as carmustine, lomustine, vincristine, procarbazine, carboplatin, cis-platin, etoposide, irinotecan, and its active metabolites, and related agents.
Concurrent administration of temozolomide (TMZ) and radiotherapy (RT) has emerged as the primary ‘standard of care’ for patients with newly diagnosed GBM. A clinically-meaningful improvement in survival compared to RT alone has been demonstrated, but the increase is still disappointing (median survival time for patients treated with TMZ/RT is 15 months, vs. only 12 months for patients treated with RT alone).
In spite of the successful introduction of TMZ-based combination therapy, however, clinicians still concur that there remains a significant need for the development of new chemotherapeutically-active agents for use in the treatment of glioma, and particularly for GBM and advanced stages of the disease. Similarly, there remains a significant, unmet need in the medical arts for new chemotherapeutic agents effective in the prevention, treatment, and/or amelioration of one or more symptoms of hyperproliferative disorders, and particularly for aggressive forms of mammalian cancers, such as human gliomal tumors.
Primary brain tumors are classified into more than 10 types according to their origin of onset and pathological tissue type, examples of which include glioma and meningioma. Gliomas are particularly serious in terms of both incidence and malignancy, and are classified into seven or more types such as glioblastoma and anaplastic astrocytoma according to their detailed pathological tissue type. Disease stage (i.e., tumor size, presence of distal metastasis) and histological malignancy are used to determine the degree of malignancy of primary brain tumors, with histological malignancy being classified into four levels of advancing degree of malignancy (G1 to G4). For example, the malignancy of glioblastoma is G4 (WHO4), while the malignancy of anaplastic astrocytoma is G3 (WHO3), and both G3 and G4 are classified as malignant. Thus, those primary brain tumors that should first be targeted by anti-brain tumor agents are gliomas, and particularly glioblastoma or anaplastic astrocytoma associated with a high degree of malignancy.
Although definitive efficacy of chemotherapy has only been confirmed for alkylating agents and temozolomide, their efficacy is limited to concomitant use with radiotherapy. Post-surgical radiotherapy has also been demonstrated to demonstrate life-prolonging (albeit brief) effects.
Therefore, an important object of the present invention is to provide novel compounds (and pharmaceutical that include them) for use in chemotherapeutic methods aimed at treating malignant cancer in affected animals, and in particular, in mammals such as humans, diagnosed with one or more forms of glioma.